Hsp90 is a molecular chaperone responsible for folding nascent polypeptides into their biologically active, three-dimensional conformations. Disruption of the Hsp90 protein folding process results in the simultaneous inhibition of multiple enzymes that are essential for malignant cell growth. In fact, proteins represented in all six hallmarks of cancer are dependent on Hsp90 for conformational activation, and several of these Hsp90 client proteins are individually sought after cancer chemotherapeutic targets. Consequently, Hsp90 inhibition offers a promising new target for the development of anticancer chemotherapeutic agents because multiple signaling pathways can be simultaneously inhibited by disruption of the Hsp90 protein folding machinery. This application aims to develop new high-throughput assays for the identification of new lead compounds that inhibit Hsp90. A cell lysate assay will be developed to screen for inhibitors that prevent the renaturation of a well-studied Hsp90 client protein that has excellent bio-luminescence properties. It is proposed that molecules capable of inhibiting this renaturation process will be subsequently analyzed for their method of Hsp90 inhibition by two independent assays aimed at identifying both N- and C-terminal ATP binding site inhibitors. Alternatively, each of these assays should be independently capable of identifying new inhibitors of Hsp90 in a complimentary manner. At the completion of this project, we expect to provide three independent assays for high-throughput detection and evaluation of inhibitors that disrupt the Hsp90 protein folding process. The therapeutic potential of these compounds could be immense, as subsequent modification of these lead compounds should lead to the development of novel high-affinity Hsp90 antagonists for the treatment of cancer.